Although extensive evidence supports the role of β-amyloid (Aβ) in Alzheimer disease (AD), the neurotoxic mechanisms underlying AD pathogenesis are not understood. On the other hand, neuronal loss is the pathologic feature that best correlates with cognitive impairment. We hypothesized that cholinergic neurodegeneration may lead to Aβ deposition and tested this by inducing selective cholinergic lesions in APPswe/PS1dE9 mice with murine p75 saporin (mu p75-SAP). Intracerebroventricular lesions that removed approximately 50% of cholinergic innervation to the cortex and hippocampus were induced in animals with incipient (∼3 months) and marked (∼7 months of age) Aβ deposition. Cranial windows were implanted, and Aβ deposition was monitored in vivo using multiphoton microscopy. Deposition of Aβ was increased as soon as 7 days after the lesion, and this effect was maintained up to 3 months later. Postmortem studies using immunohistochemistry with an anti-Aβ antibody corroborated these findings in both cerebral cortex and hippocampus. Tau phosphorylation was also significantly increased after the lesions. Cholinergic denervation resulted in early memory impairment at 3 months of age that worsened with age (∼7 months); there was a synergistic effect between cholinergic denervation and the presence of APP/PS1 transgenes. Altogether, our data suggest that cholinergic denervation may trigger Aβ deposition and synergistically contribute to cognitive impairment in AD patients.